CN101503718A - Preparation of Tetramethylpyrazine - Google Patents
Preparation of Tetramethylpyrazine Download PDFInfo
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- CN101503718A CN101503718A CNA2009100968149A CN200910096814A CN101503718A CN 101503718 A CN101503718 A CN 101503718A CN A2009100968149 A CNA2009100968149 A CN A2009100968149A CN 200910096814 A CN200910096814 A CN 200910096814A CN 101503718 A CN101503718 A CN 101503718A
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Abstract
The invention discloses a method for preparing ligustrazine. The method comprises the following steps that: an LB inclined plane seed of the bacillus subtilis natto is cultured and transferred to an LB liquid culture medium for culture to obtain seed culture solution; the seed culture solution is inoculated to a soybean culture medium for culture and conversion at a temperature of between 30 and 40 DEG C for 144 to 168 hours to obtain a ligustrazine containing product; and the ligustrazine containing product is added with organic solvent for extraction, the obtained extracting solution is evenly mixed with diluted hydrochloric acid and is stood for layering, and the water phase is the water solution of hydrochloric ligustrazine. The method has the advantages of simple operation, easy culture of microbial cells, easy control of biological conversion, high safety, no pollution, and suitability for industrialized production.
Description
Technical field
The present invention relates to bioengineering field and food chemistry field, be specifically related to a kind of method of utilizing Bacillus subtilis natto to prepare Ligustrazine.
Background technology
Ligusticum wallichii, the dry rhizome of samphire Ligusticum wallichii (Ligusticum chuanxiong Hort.).Also Cheng Xiong is per nnial herb, root stock, tawny; Two or three times pinnately compound leaves, 3 to 5 pairs on leaflet, the edge becomes irregular pinniform to totally cleave or drastic crack; Floral white, compound umbel; Mericarp is avette, and sharp rib is arranged.Root stock is used as medicine, and is warm in nature, flavor is hot, invigorates blood circulation, promoting the circulation of qi, menstruation regulating, the functions such as the stasis of blood, pain relieving of dispeling the wind, dispel, and cures mainly headache, menoxenia, through closing diseases such as dysmenorrhoea, treating swelling and pain by traumatic injury, rheumatic arthralgia, is used for the treatment of coronary heart disease, acute and chronic ischemic cerebrovascular disease.
Ligusticum wallichii is the traditional Chinese medicinal materials of China, and main chemical compositions has acid in forulic acid, Ligustrazine, chuanxingol, perlolyrine, ligustilide, the Ligusticum wallichii phthalein, also contains chemical substances such as multiple organic acid, ester, alcohol, sugar, vitamin A and trace element etc.Ligustrazine (Ligustrazine, Lig; Tetramethylpyrazine is a kind of active alkaloid that extracts the root, stem from umbrella section plant Ligusticum wallichii TMP), and formal name used at school is a Tetramethylpyrazine.The Ligustrazine molecular formula is C
8H
12N
2, relative molecular mass is 136.20; Outward appearance is colourless needle, and special odor is arranged, and fusing point is 80～82 ℃, and boiling point is 190 ℃, the moisture absorption, easily distillation; Be dissolved in hot water, sherwood oil, chloroform, dilute hydrochloric acid, be slightly soluble in ether, be insoluble in cold water.
People have all done deep research to the pharmacology and the clinical application of Ligustrazine in recent years, according to animal pharmacology experiment and observation on Clinical Application, Ligustrazine has pharmacological actions such as obvious coronary artery dilator, coronary blood flow increasing, lax vascular smooth muscle, reduction surface activity of blood platelet.Can be used for improving stenocardia and myocardial ischemia clinically, treatment ischemia cerebral thrombosis and cerebral infarction, the antagonism calcium ion, reduce blood viscosity, strengthen myocardial contraction, to correct heart failure, relaxing smooth muscle, reduce glandular secretion, microcirculation improvement, diseases such as treatment ephritis, diabetes, ephrosis, interstitial lung fiber, infantile asthma and bronchitis, facial paralysis, deafness, sacroiliitis.
Ligustrazine mainly is to extract to obtain from the Chinese medicinal materials Ligusticum wallichii at present, Zhao Huiping has carried out studying (Chinese medicine collection to the extraction process of Ligusticum wallichii, 2004,4 10 phases of volume, the Ligusticum wallichii Study on extraction process), adopting four kinds of extracting method to draw the optimum extraction scheme, prove under acidic conditions, is feasible with the technology of ethanol-extracted Ligustrazine.Human fado rope solvent, supercritical COs such as Liu Ben
2Or contain the supercritical CO of 10% methyl alcohol
2Extract Ligustrazine (Chinese Journal of Pharmaceuticals,, 2 30 phases of volume, the Ligustrazine in fado rope solvent and the supercritical extraction Ligusticum wallichii in 1999) from Ligusticum wallichii, the HPLC analytical results shows, through 2 hours extraction, contains the supercritical CO of 10% methyl alcohol
2Extraction yield is the highest by 1.02%, and fado rope solvent is through 3 times 1.5 hours extraction, extraction yield minimum 0.74%.
Ligustrazine also can adopt the method for chemosynthesis to prepare, a kind of preparation method of Tetramethylpyrazine is disclosed among the Chinese patent CN1100765C, in the presence of the coordination catalyst of VIII family metallic compound and organic ligand formation, by hydrogen and 2,3-dimethyl diketone monoxime reacts and obtains Tetramethylpyrazine, and wherein having investigated differential responses temperature, hydrogen partial pressure, reaction times, solvent etc. influences rule to its synthetic.Chinese patent CN1238344C discloses a kind of production method of Tetramethylpyrazine, is raw material with amino butanone, feeds steam under alkaline condition, and the Tetramethylpyrazine that obtains can be trihydrate forms.Though above-mentioned chemical synthesis productive rate is higher, is easy to generate many by products, also can bring environmental pollution and safety issue.
Bio-transformation is to utilize plant isolated cells or organ, zooblast, microorganism and organoid thereof, and resolvase carries out the biochemical reaction of structural modification to exogenous compounds.Wherein microorganism though its volume is little, also has the enzyme system of a cover autospecific as a complete individuality, and aboundresources, of a great variety, for special substrate, and can be a lot of for the bacterial strain of screening.Utilize the enzyme system of multiple different catalysiss that active skull cap components is carried out bio-transformation at present, produce the natural compounds storehouse of new associativity, pass through screening active ingredients again, seek the natural radioactivity lead compound of new high-efficiency low-toxicity, or, huge social benefit and economic benefit have been obtained by the analysis of heterogeneity structural changes in the active ingredient and activity intensity growth and decline relation being found the lead compound of a new generation.
Utilizing the process of conversion technology developing new drug, is to be lead compound with the natural product, by chemistry or biological means, its structure is modified, and obtains the compound of the many novel structures of root, and then finds that toxicity is lower, the medicine of better efficacy.As seen the structural modification of new drug is being instructed in bio-transformation, the importance in the medicine of new generation of acquisition efficient long-acting.
Summary of the invention
The invention provides a kind of preparation method of Ligustrazine, utilize Bacillus subtilis natto that soybean is carried out bio-transformation and prepare Ligustrazine, this method is simple to operate, and conversion reaction is easy to control, and is pollution-free, safe.
A kind of preparation method of Ligustrazine comprises:
(1) will be transferred to the LB liquid nutrient medium after the Bacillus subtilis natto LB inclined-plane seed culture, cultivate at 30～40 ℃ (preferred 37 ℃) and obtained seed culture fluid in 18～24 hours;
Described Bacillus subtilis natto is the bacterial classification commonly used that Japan produces natto, can adopt the commercially available prod;
The density of bacterium cell is 1 * 10 in the seed culture fluid that obtains
7～1 * 10
9Cfu/ml.
(2) seed culture fluid that step (1) is obtained inserts soya broth, cultivates conversion promptly obtains containing Ligustrazine after 144～168 hours product at 30～40 ℃ (preferred 37 ℃);
Described soya broth is to obtain after the soybean sterilising treatment of will soak with flowing water, and the sterilising treatment process is conventional high-temperature sterilization, preferably 115 ℃ of sterilising treatment 25 minutes.
The weight ratio of seed culture fluid and soya broth is preferably 0.5～8: 100, be preferably 1: 100.
Cultivate in the conversion process and can add an amount of acetoin, the interpolation time of acetoin can be chosen in to cultivate and transform when carrying out 0～132 hour, preferably when the cultivation conversion is carried out 96 hours; The addition of acetoin is 0.1%～3%, preferred 0.5% of nutrient solution and a soya broth gross weight.
Show that after deliberation the adding of acetoin can significantly improve transformation efficiency, the purity of target product is also had good improvement.
(3) the adding organic solvent extracts and obtains extracting solution in the product that contains Ligustrazine that step (2) obtains, and extracting solution and dilute hydrochloric acid are mixed the back standing demix, and water is the Muriatic Ligustrazine aqueous solution.
The consumption that contains the product of Ligustrazine and organic solvent can preferably contain the weight (g) of the product of Ligustrazine: volume of organic solvent (mL)=1: 5～50, preferred 1: 25; Guaranteeing fully to extract Ligustrazine, and save organic solvent.
Described organic solvent is that methyl alcohol, acetone, ethyl acetate, the 1mol/L NaOH aqueous solution and chloroform volume ratio are 1: 1 mixing solutions or chloroform.
Described organic solvent can also be the mixing solutions of acetate, second alcohol and water, and its weight percent consists of acetate 5%, ethanol 65%～80%, and surplus is a water.Adopt this mixing solutions,, can obviously improve the yield of extraction because the polarity of its polarity and purpose product is approaching.
Generally can adopt pre-configured aqueous ethanolic solution to mix during the configuration of this mixing solutions with an amount of acetate.
Utilize the process of organic solvent extraction can be chosen under the ultrasonic wave auxiliary treatment, remove by filter residue, obtain extracting solution in 20～60 ℃ of refluxing extraction 0.5～4 hour;
Hyperacoustic power can be selected 220～880W, extracts temperature and can be 23～57 ℃, and extraction time is 0.5～3 hour.
Described dilute hydrochloric acid concentration is 0.1～0.4mol/L, preferred 0.2～0.25mol/L.
Contain Ligustrazine in the Muriatic Ligustrazine aqueous solution, can directly utilize or use as required the prior art means and concentrate or purify.
Carry out reverse high performance liquid chromatography (RP-HPLC) after the Muriatic Ligustrazine aqueous solution is handled after filtration and detect, testing conditions is: moving phase: mass concentration is the volume ratio=45: 55 of 1% aqueous acetic acid and methyl alcohol; Flow velocity: 0.8mL/min; Detect wavelength: 290nm; Column temperature: 45 ℃; Sample size: 2 μ L.
The preparation method of Ligustrazine of the present invention has utilized the characteristic of Bacillus subtilis natto, and operating process is simple, and microorganism cells is cultivated easily, and bio-transformation is easy to control, and is safe, and non-environmental-pollution, is suitable for suitability for industrialized production.
Embodiment
Embodiment 1
Bacillus subtilis natto LB inclined-plane seed culture is transferred to the LB liquid nutrient medium after 24 hours, in 37 ℃, 150r/min shaking table, cultivates and obtained seed culture fluid in 18 hours.
Take by weighing the soybean that 100g soaks with flowing water and add in the 500mL triangular flask,, obtain required substratum in 115 ℃ of sterilising treatment 25 minutes.
With the above-mentioned substratum of the aseptic access of 2g seed culture fluid, cultivate conversion for 37 ℃ and promptly obtain the product that 102g contains Ligustrazine after 144 hours, add the 510mL chloroform, at power is under the ultrasonic wave auxiliary treatment of 550W, remove residue in 1.5 hours after-filtration of 30 ℃ of refluxing extraction, obtain extracting solution, the dilute hydrochloric acid of the 0.2mol/L of adding and extracting solution equal volume, mix the back standing demix, water is the Muriatic Ligustrazine aqueous solution.
The above-mentioned Muriatic Ligustrazine aqueous solution is carried out RP-HPLC detect, detected result is: every 100g soybean can transform and make the 0.0214mg Ligustrazine.
Embodiment 2
Bacillus subtilis natto LB inclined-plane seed culture is transferred to the LB liquid nutrient medium after 24 hours, in 37 ℃, 150r/min shaking table, cultivates and obtained seed culture fluid in 18 hours.
Take by weighing the soybean that 100g soaks with flowing water and add in the 500mL triangular flask,, obtain required substratum in 115 ℃ of following sterilising treatment 25 minutes.
With the above-mentioned substratum of the aseptic access of 2g seed culture fluid, cultivate conversion in 37 ℃, cultivate and add the 0.2g acetoin when transforming 96 hours, cultivate conversion and promptly obtain the product that 102.2g contains Ligustrazine after 144 hours, add 511mL acetone, at power is under the ultrasonic wave auxiliary treatment of 550W, remove residue in 1.5 hours after-filtration of 43 ℃ of refluxing extraction, obtain extracting solution, the dilute hydrochloric acid of the 0.2mol/L of adding and extracting solution equal volume, mix the back standing demix, water is the Muriatic Ligustrazine aqueous solution.
The above-mentioned Muriatic Ligustrazine aqueous solution is carried out RP-HPLC detect, detected result is: every 100g soybean can transform and make the 0.698mg Ligustrazine.
Embodiment 3
Bacillus subtilis natto LB inclined-plane seed culture is transferred to the LB liquid nutrient medium after 24 hours, in 37 ℃, 150r/min shaking table, cultivates and obtained seed culture fluid in 18 hours.
Take by weighing the soybean that 100g soaks with flowing water and add in the 500mL triangular flask,, obtain required substratum in 115 ℃ of following sterilising treatment 25 minutes.
With the above-mentioned substratum of the aseptic access of 5g seed culture fluid, under 37 ℃, cultivate conversion, cultivate and add the 0.2g acetoin when transforming 96 hours, cultivate conversion and promptly obtain the product that 105.2g contains Ligustrazine after 144 hours, add the 5260mL chloroform, at power is under the ultrasonic wave auxiliary treatment of 880W, remove residue in 2.5 hours after-filtration of 25 ℃ of refluxing extraction, obtain extracting solution, the dilute hydrochloric acid of the 0.2mol/L of adding and extracting solution equal volume, mix the back standing demix, water is the Muriatic Ligustrazine aqueous solution.
The above-mentioned Muriatic Ligustrazine aqueous solution is carried out RP-HPLC detect, detected result is: every 100g soybean can transform and make the 0.888mg Ligustrazine.
Embodiment 4
Bacillus subtilis natto LB inclined-plane seed culture is transferred to the LB liquid nutrient medium after 24 hours, in 37 ℃, 150r/min shaking table, cultivates and obtained seed culture fluid in 18 hours.
Take by weighing the soybean that 100g soaks with flowing water and add in the 500mL triangular flask,, obtain required substratum in 115 ℃ of following sterilising treatment 25 minutes.
With the above-mentioned substratum of the aseptic access of 1g seed culture fluid, under 37 ℃, cultivate conversion, cultivate and add the 0.48g acetoin when transforming 96 hours, cultivate conversion and promptly obtain the product that 101.48g contains Ligustrazine after 168 hours, the mixing solutions 2537ml that adds acetate, second alcohol and water extracts, the mixing solutions weight percent consists of acetate 5%, ethanol 70%, and surplus is a water.At power is under the ultrasonic wave auxiliary treatment of 880W, remove residue in 2.5 hours after-filtration of 25 ℃ of refluxing extraction, obtain extracting solution, the dilute hydrochloric acid of the 0.25mol/L of adding and extracting solution equal volume, mix the back standing demix, water is the Muriatic Ligustrazine aqueous solution.
The above-mentioned Muriatic Ligustrazine aqueous solution is carried out RP-HPLC detect, detected result is: every 100g soybean can transform and make the 0.92mg Ligustrazine.
Claims (10)
1, a kind of preparation method of Ligustrazine comprises:
(1) will be transferred to the LB liquid nutrient medium after the Bacillus subtilis natto LB inclined-plane seed culture, cultivate at 30～40 ℃ and obtained seed culture fluid in 18～24 hours;
(2) seed culture fluid that step (1) is obtained inserts soya broth, cultivates conversion promptly obtains containing Ligustrazine after 144～168 hours product at 30～40 ℃;
(3) in the product that contains Ligustrazine that step (2) obtains, add organic solvent and extract, obtain extracting solution, extracting solution is isolated water after with the dilute hydrochloric acid acidifying, obtain the Muriatic Ligustrazine aqueous solution.
2, preparation method as claimed in claim 1 is characterized in that: in the step (1), the density of bacterium cell is 1 * 10 in the seed culture fluid
7～1 * 10
9Cfu/mL.
3, preparation method as claimed in claim 1 is characterized in that: in the step (2), the weight ratio of seed culture fluid and soya broth is 0.5～8: 100.
4, preparation method as claimed in claim 1 is characterized in that: in the step (2), add acetoin in cultivating conversion process.
5, preparation method as claimed in claim 4 is characterized in that: the interpolation time of described acetoin, addition was 0.1%～3% of seed culture fluid and a soya broth gross weight for when the cultivation conversion is carried out 0～132 hour.
6, preparation method as claimed in claim 1 is characterized in that: organic solvent described in the step (3) is that methyl alcohol, acetone, ethyl acetate, the 1mol/L NaOH aqueous solution and chloroform volume ratio are 1: 1 mixing solutions or chloroform.
7, preparation method as claimed in claim 1 is characterized in that: organic solvent is the mixing solutions of acetate, second alcohol and water described in the step (3), and its weight percent consists of acetate 5%, ethanol 65%～80%, and surplus is a water.
8, preparation method as claimed in claim 1 is characterized in that: described leaching process under the ultrasonic wave auxiliary treatment in 20～60 ℃ of refluxing extraction 0.5～4 hour, remove by filter residue, obtain extracting solution.
9, preparation method as claimed in claim 1 is characterized in that: the concentration of described dilute hydrochloric acid is 0.1～0.4mol/L.
10, preparation method as claimed in claim 9 is characterized in that: the concentration of described dilute hydrochloric acid is 0.2～0.25mol/L.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2009100968149A CN101503718B (en) | 2009-03-16 | 2009-03-16 | Preparation of Tetramethylpyrazine |
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| Application Number | Priority Date | Filing Date | Title |
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| CN2009100968149A CN101503718B (en) | 2009-03-16 | 2009-03-16 | Preparation of Tetramethylpyrazine |
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| CN101503718A true CN101503718A (en) | 2009-08-12 |
| CN101503718B CN101503718B (en) | 2012-05-09 |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101906448B (en) * | 2010-02-02 | 2012-07-11 | 浙江大学 | Method for biosynthesizing ligustrazine |
| CN106083744A (en) * | 2016-06-23 | 2016-11-09 | 黄增琼 | A kind of method from Semen Podocarpi Macrophylli seed high efficiency extraction separating high-purity ligustrazine |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1513845A (en) * | 2003-08-18 | 2004-07-21 | 淄博正存化工有限公司 | Separation and purification method of 2,5-dimethyl pyrazine |
-
2009
- 2009-03-16 CN CN2009100968149A patent/CN101503718B/en not_active Expired - Fee Related
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101906448B (en) * | 2010-02-02 | 2012-07-11 | 浙江大学 | Method for biosynthesizing ligustrazine |
| CN106083744A (en) * | 2016-06-23 | 2016-11-09 | 黄增琼 | A kind of method from Semen Podocarpi Macrophylli seed high efficiency extraction separating high-purity ligustrazine |
| CN106083744B (en) * | 2016-06-23 | 2018-08-14 | 黄增琼 | A method of from podocarpus seed high efficiency extraction separating high-purity ligustrazine |
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| CN101503718B (en) | 2012-05-09 |
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